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P53 protein monoclonal antibody and application thereof

A monoclonal antibody and protein technology, applied in the field of biological sciences, can solve the problems of difficult antibody preparation and screening methods, and the detection effect of different tumor samples needs to be improved, so as to achieve the effect of strong specific recognition effect and strong positive signal

Pending Publication Date: 2022-04-08
武汉赛维尔生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the process of antibody preparation, exogenously expressed protein fragments or chemically synthesized short peptides are usually selected as immunogens through coupling carriers, and the target antibodies are obtained through immunization of animals and final detection. Immunogen fragments need to undergo antigenicity, hydrophilicity, Surface analysis, etc., not all fragments can successfully prepare antibodies, and the specificity of antibodies prepared by different immunogen fragments is also different, and it is not easy to obtain effective antibodies by a single preparation and screening method
[0004] There are many p53 antibodies on the market, and the published immunogen sequences are also inconsistent. However, considering the actual effect, only DO-7 is the most commonly used p53 commercial antibody that can be used for diagnosis, but its detection effect on different tumor samples needs to be improved

Method used

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  • P53 protein monoclonal antibody and application thereof
  • P53 protein monoclonal antibody and application thereof
  • P53 protein monoclonal antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] The preparation of embodiment 1 immunogen

[0039] 1. Construction of p53 recombinant expression vector

[0040] By means of conventional gene synthesis, according to the amino acid composition, antigenicity, hydrophilicity and hydrophobicity of the sequence, the secondary structure and the specificity of the sequence, etc., the 352nd to 352nd to the p53 protein encoding the amino acid sequence shown in SEQ ID NO: 7 were selected. The gene sequences of amino acids 366, 1-131, 99-112, 295-376, 1-80 and 15-39 were inserted into the E.coil pet32a vector by restriction endonucleases EcoRI and xhoI, respectively. Construct the recombinant expression plasmid of p53. The amino acid sequence of human p53 protein was obtained from the Uniprot database (https: / / www.uniprot.org / uniprot / P04637#sequences).

[0041] 2. Expression and purification of p53 recombinant protein

[0042] The p53 recombinant expression plasmid constructed in step 1 was transformed into Escherichia coli R...

Embodiment 2

[0043] The preparation of embodiment 2 polypeptide

[0044] The method of chemical synthesis synthesizes polypeptides with amino acid sequences as shown in SEQ ID NO: 1-6, and synthesizes one more cysteine ​​(Cyc) at the N-terminus of the polypeptides to obtain polypeptides 1 to 6 for coating enzyme labels Version.

Embodiment 3

[0045] Embodiment 3 immune mice and carry out ELISA screening

[0046] The p53 recombinant protein 5 purified in Example 1 (amino acid sequence shown in SEQ ID NO: 5) was used to immunize female balb / c mice aged 6 to 8 weeks, with an interval of 3 weeks between the first immunization and booster immunization, and subsequent booster immunization The interval was 2 weeks, at least 4 times of immunization, the spleen was taken to make a single cell suspension, and the SP2 / 0 cells in the logarithmic growth phase were taken at the same time 1×10 7 One, using the commercial reagent PEG1450 (Sigma, P7181) for cell fusion, uniformly dilute the fused hybridoma cells into a certain volume of 1% HAT (Sigma, H0262) selection medium, and plate 96-well cell culture plates for 6 ~8 pieces, 200 μL / well, after static culture for 4 to 6 days, visible hybridoma cell colonies are formed, and the liquid is changed to obtain the supernatant on the fusion plate, which is to be tested.

[0047] Use ...

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Abstract

The invention discloses a p53 protein monoclonal antibody and application thereof. The invention discloses an immunogen fragment of a p53 protein monoclonal antibody. The fragment is a p53 protein 1-80 region; a corresponding coating antigen peptide fragment screened by ELISA is a p53 protein 15-39 region, and one more cysteine is synthesized at the N end of the polypeptide. The cloning number of the monoclonal cell strain is 4H12E8, the subtype of a secreted antibody is IgG1, and the cell strain can secrete a p53 protein monoclonal antibody. Compared with an immunogen and a coating antigen, the p53 protein monoclonal antibody disclosed by the invention has the same peptide fragment, the prepared p53 protein monoclonal antibody has stronger specific recognition effect, can recognize p53 proteins in various samples, and has stronger positive signal and superior positive rate compared with a commercially common p53 protein monoclonal antibody; the method is suitable for detecting p53 protein in various tumor samples.

Description

technical field [0001] The invention relates to the technical field of biological sciences, in particular to a p53 protein monoclonal antibody and its application. Background technique [0002] p53 is a tumor suppressor gene, which ensures the repair of damaged DNA before cell division. It is an indicator of poor prognosis in most tumors. It is divided into wild type and mutant type. Its molecular weight is 53kD, so it is called p53 protein; wild type P53 protein is extremely unstable, with a short half-life, which cannot be detected by immunohistochemistry. The stability of mutant p53 protein is increased, the half-life is prolonged, and it accumulates in the nucleus, which can be detected by immunohistochemistry. p53 is so far found to be most relevant to human tumors The gene has been widely used in the diagnosis of tumors; [0003] The main way of loss of p53 normal function is gene mutation. Through the analysis of a large number of mutants in tumors, it has been confi...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/18G01N33/574G01N33/577
Inventor 郑沣马婷万新春胡杨王绪德赵红洲
Owner 武汉赛维尔生物科技有限公司
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